As used herein, the term “angiogenesis” means the generation of new blood vessels into a tissue or organ. Under normal physiological conditions, humans or animals undergo angiogenesis only in very specific restricted situations. For example, angiogenesis is normally observed in wound healing, fetal and embryonal development and formation of the corpus luteum, endometrium and placenta. The term “endothelium” means a thin layer of flat epithelial cells that lines serous cavities, lymph vessels, and blood vessels. The term “endothelial modulating activity” means the capability of a molecule to modulate angiogenesis in general and, for example, to stimulate or inhibit the growth of endothelial cells in culture. Both controlled and uncontrolled angiogenesis are thought to proceed in a similar manner. Endothelial cells and pericytes, surrounded by a basement membrane, form capillary blood vessels. Angiogenesis begins with the erosion of the basement membrane by enzymes released by endothelial cells and leukocytes. The endothelial cells, which line the lumen of blood vessels, then protrude through the basement membrane. Angiogenic stimulants induce the endothelial cells to migrate through the eroded basement membrane. The migrating cells form a “sprout” off the parent blood vessel, where the endothelial cells undergo mitosis and proliferate. The endothelial sprouts merge with each other to form capillary loops, creating the new blood vessel.
Persistent, unregulated angiogenesis occurs in a multiplicity of disease states, and abnormal growth by endothelial cells and supports the pathological damage seen in these conditions. The diverse pathological disease states in which unregulated angiogenesis is present have been grouped together as angiogenic dependent or angiogenic associated diseases.
It is also recognized that angiogenesis plays a major role in the metastasis of a cancer. If this angiogenic activity could be repressed or eliminated, then the tumor, although present, would not grow. In the disease state, prevention of angiogenesis could avert the damage caused by the invasion of the new microvascular system. Therapies directed at control of the angiogenic processes could lead to the abrogation or mitigation of these diseases.
The development of renal glomerular capillaries is anatomically segregated and temporally staged in a multi-step process. The process involves recruitment of endothelial progenitors from adjacent mesenchyme, assembly of an arborized branching network, and maturation and specialization of endothelial cells adjacent to mesangial and visceral epithelial cells. Receptors for extracellular matrix components, cell surface molecules and growth factors have been assigned roles to mediate steps in this assembly process. See e.g., Wallner et al., Microsc Res Tech 39:261–284 (1997); Takahashi et al., Kidney Int 53:826–835 (1998).
Vascular endothelial growth factor (VEGF) is an important participant, as it is induced in S stage developing glomerular epithelial cells, and endothelial progenitors that are recruited to glomerular capillaries from the adjacent metanephric mesenchyme express the VEGF receptor, flk-1. Robert et al., Am J Physiol 271:F744–F753 (1996).
Neutralizing VEGF antibodies interrupt postnatal murine glomerular capillary development. Kitamoto et al., J Clin Invest 99:2351–2357 (1997). Deletion of either PDGFβ receptor or PDGFβ genes in mice causes defective recruitment of mesangial cell precursors with failure of glomerular development. Soriano, P., Genes Dev 8:1888–1896 (1994); Leveen et al., Genes Dev 8:1875–1887 (1994). TGFβ1 expression and type II TGFβ receptors appear critical for vascular development in the embryonic yolk sac (prior to renal development), and type II receptors mediate in vitro capillary morphogenesis of endothelial cells derived from bovine glomeruli. Choime et al., J Biol Chem 270:21144–21150 (1995).
Early evidence suggests that Eph family receptors and their ephrin ligands participate in glomerular vascular development. EphB1 receptors are expressed in isolated mesenchymal cells in a pattern similar to that of flk-1, and high level expression of ephrin-B1 is seen at the vascular cleft of developing glomeruli, as well as in capillary endothelial cells of mature glomeruli. Daniel et al., Kidney Int 50:S-73–S-81 (1996). Oligomerized forms of ephrin-B1 stimulate in vitro assembly of human renal microvascular endothelial cells (HRMEC) into capillary-like structures. Stein et al., Genes Dev 12:667–678 (1998).
A selected subclass of receptor tyrosine phosphatases, including DPTP10D, serve important roles in directing axonal migration and neural network assembly. Desai et al., Cell 84:599–609 (1996). Recent data has identified mRNA expression of a related receptor phosphatase, ECRTP/DEP-1, in arterial sites in mammalian kidney. Borges et al., Circulation Research 79:570–580 (1996). To date, however, there has been no evidence to implicate receptor tyrosine phosphatases in microvascular or glomerular capillary assembly or maturation.
Vascular endothelial cells display a diverse range of vascular bed specific properties (Gumkowski et al., Blood Vessels 24:11–13 (1987)), yet the requirement to maintain a continuous, antithrombotic monolayer lining the vascular space imposes rigorous requirements that their proliferation, migration and differentiation be regulated by interendothelial contacts. Specialized intercellular contacts permit communication among interacting endothelial cells (Lampugnani et al., J Cell Biol 129:203–217 (1995)) yet the mechanisms regulating arrest of proliferation and migration in response to interendothelial contact have not been elucidated. Tight regulatory control over proliferation imposed by interendothelial cell contact is apparent in the low basal mitotic index among endothelial cells in existing vessels. Engerman et al., Laboratory Investigation 17:738–744 (1967). This is in contrast with the proliferative endothelial responses that are evoked by mechanical disruption of large vessels. More et al., J Patho 172:287–292 (1994). Similar proliferation and migration responses are stimulated at the margin of a confluent endothelial monolayer by “wounding”, or physical removal cells from the packed monolayer. Coomber, J Cell Biochem 52:289–296 (1993).
The molecular basis for effects of interendothelial contact on migratory and proliferative responses is not defined, yet studies of cultured cells have shown that endothelial, fibroblast, and epithelial cells grow to confluency at a predictable density, then arrest proliferation (density arrest). Augenlicht and Baserga, Exp Cell Res 89:255–262 (1974); Beekhuizen and van Furth, J Vascular Res 31:230–239 (1994); Rijksen et al., J Cell Physiol 154:393–401 (1993). This phenomenon can be very relevant to the behavior of endothelial cells in vascular sites in situ. Indeed, model culture systems of endothelial “wounding” have shown that endothelial cells at the edge of an imposed “wound” rapidly extend lamellae, spread, migrate and proliferate to replace the deficit created by mechanical disruption of the monolayer. Coomber, J Cell Biochem 52:289–296 (1993).
Pallen and Tong observed that membrane-associated tyrosine phosphatase activity recovered from cultured Swiss 3T3 cells increased eight (8)-fold (expressed as activity/mg protein) as cells approached a density of 5×104/cm2, while soluble fraction tyrosine phosphatase was unaffected by cell density. Pallen and Tong, Proc Natl Acad Sci USA 88:6996–7000 (1991). Ostman et al. determined that the abundance of a receptor tyrosine phosphatase cloned from HeLa cells and named DEP-1, is increased as cells approach high density. Ostman et al., Proc Natl Acad Sci USA 91:9680–9684 (1994). However, no links between molecules that evoke proliferation arrest and receptor tyrosine phosphatases have been made.
To date, available information does not indicate what sort of receptor-ligand interaction might mediate a cell surface generated signal for density or contact arrest. The identification of such a receptor-ligand interaction is therefore needed in that it will serve as a basis for intervention in a disorder wherein density or contact arrest, or the preclusion of density or contact arrest, has therapeutic value. Such disorders include disorders characterized by undesired angiogenesis, such as angiogenesis associated with tumor growth. Thus, what is also needed is a composition and method which can inhibit the unwanted growth of blood vessels, especially into tumors. The composition and method should attenuate the formation of the capillaries in the tumors thereby inhibiting the growth of the tumors.